Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained.

Abstract

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard '251-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (< 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA.Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.

Prourokinase Mutant That Induces Highly Effective Clot Lysis Without Interfering With Hemostasis

“…When M5 (2 g/mL) was combined with a small amount (30 ng/mL) of tPA, which was insufficient to induce clot lysis by itself, the lag phase was reduced by half, as previously observed with similar combinations of tPA and proUK. 25 This has been ascribed to the creation of new (fragment E) plasminogen binding sites by tPA-induced lysis, which promote plasminogen activation by proUK and M5 (see Table 2). Therefore, fibrin-dependent plasminogen activation by M5 is similarly complementary to tPA (promoted by fragment D) as proUK 25 (data not shown).…”

Activated Human Plasma Carboxypeptidase B Is Retained in the Blood by Binding to α2-Macroglobulin and Pregnancy Zone Protein

“…These Lys binding sites are also important for the activation and regulation of plasminogen (Lijnen et al, 1980;Clemmensen, et al, 1981Clemmensen, et al, , 1986Pannell et al, 1988;Miles et al, 1991;Lenich et al, 1991). Specifically, the binding of ␣ 2 -antiplasmin to plasminogen have been shown to depend on Lys residues present in the COOHterminal region of ␣ 2 -antiplasmin (Sasaki et al, 1986;Hortin et al, 1988).…”

“…In particular, it has been suggested that the binding of histidine-rich glycoprotein to the Lys binding sites of plasminogen may prevent the binding of plasminogen to fibrinogen and fibrin (Lijnen et al, 1980;Ichinose et al, 1984). It is similarly conceivable that a Lys-specific carboxypeptidase may interfere with the degradation of the fibrin clot by removing COOH-terminal Lys residues from fibrin (Pannell et al, 1988;Miles et al, 1991;Bajzar et al, 1995).…”

Thrombolysis, the only Optimally Rapid Reperfusion Treatment

“…Fibrinolysis induced by such low tPA concentrations can be explained by the complementary modes of action of tPA and proUK [15] and their ibrinolytic effect which when combined is synergistic [16]. Unlike tPA that is stored in the vessel wall, from where it initiates ibrinolysis, proUK is a is a normal constituent of blood, from where it continues ibrinolysis.…”

“…In view of the complementary properties of tPA and proUK and their complementary functions in ibrinolysis [15], a logical solution for improving thrombolysis is by using a combination of both plasminogen activators, as in physiological ibrinolysis.…”